Peptide degradation: effect of substrate phosphorylation on aminopeptidasic hydrolysis

The effect of substrate phosphorylation on the susceptibility to exopeptidasic attack by leucyl aminopeptidase of swine kidney, alanyl aminopeptidase from human liver and aminopeptidase N of Escherichia coli was investigated using a synthetic heptapeptide (L-R-R-A-S-L-G) and its phosphorylated derivative. The enzyme-catalyzed products were analyzed by thin layer chromatography and electrophoresis. The sensitivities of peptide and phosphopeptide to leucyl aminopeptidase digestion were then compared. Data obtained indicated that when phosphopeptide was used as substrate one main product accumulated, which corresponded to the fragment A-S(P)-L-G, while unphosphorylated peptide was completely degraded to its constituent amino acids. Identical results were obtained using aminopeptidase N of E. coli. Using alanyl aminopeptidase as enzyme, the results obtained were essentially similar, since the exopeptidasic activity on the phosphorylated peptide was strongly hampered in the vicinity of phosphoseryl residue leading to accumulation of the same phosphorylated product, although this enzyme could not completely degrade the unphosphorylated peptide. It was concluded that phosphorylation of substrates does effect enzymic degradation of proteins.